U.S. patent application number 10/594443 was filed with the patent office on 2008-09-18 for composition having antitumor effect.
This patent application is currently assigned to Genomldea Inc.. Invention is credited to Masayuki Fukumura, Yasufumi Kaneda, Hirokazu Kawano, Hitoshi Kotani, Masayuki Kurooka.
Application Number | 20080226674 10/594443 |
Document ID | / |
Family ID | 35063530 |
Filed Date | 2008-09-18 |
United States Patent
Application |
20080226674 |
Kind Code |
A1 |
Kotani; Hitoshi ; et
al. |
September 18, 2008 |
Composition Having Antitumor Effect
Abstract
The present invention is intended to provide a pharmaceutical
composition for delivering a chemotherapeutic, preferably an
anticancer drug, into cells or into a living organism, using a
viral envelope vector, and provides a pharmaceutical composition
comprising a chemotherapeutic encapsulated in, or used in
combination with, a viral envelope vector having an adjuvanticity
as an active ingredient. Thereby it is possible to introduce an
anticancer drug encapsulated in a viral envelope vector directly
into a tumor, with coadministration of another anticancer drug so
as to induce tumor cell-specific antitumor immunity also thanks to
the adjuvant action of HVJ-E, and hence to regress the tumor. The
present invention also provides a pharmaceutical composition
comprising a viral envelope vector and a chemotherapeutic as active
ingredients.
Inventors: |
Kotani; Hitoshi; (Hyogo,
JP) ; Kaneda; Yasufumi; (Osaka, JP) ; Kawano;
Hirokazu; (Osaka, JP) ; Fukumura; Masayuki;
(Osaka, JP) ; Kurooka; Masayuki; (Osaka,
JP) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
1100 13th STREET, N.W., SUITE 1200
WASHINGTON
DC
20005-4051
US
|
Assignee: |
Genomldea Inc.
Osaka
JP
AnGesMG, Inc.
Osaka
JP
|
Family ID: |
35063530 |
Appl. No.: |
10/594443 |
Filed: |
March 31, 2005 |
PCT Filed: |
March 31, 2005 |
PCT NO: |
PCT/JP2005/006820 |
371 Date: |
December 20, 2006 |
Current U.S.
Class: |
424/207.1 ;
424/204.1; 424/229.1; 424/232.1; 424/233.1 |
Current CPC
Class: |
A61K 31/24 20130101;
A61K 31/7064 20130101; A61P 35/00 20180101; A61K 2039/6006
20130101; A61K 31/704 20130101; A61K 2039/585 20130101; C12N
2760/18842 20130101; A61K 9/5184 20130101; A61K 39/0011 20130101;
A61K 2039/55572 20130101; A61K 2039/55561 20130101; A61K 31/282
20130101; A61P 37/04 20180101 |
Class at
Publication: |
424/207.1 ;
424/204.1; 424/233.1; 424/229.1; 424/232.1 |
International
Class: |
A61K 39/21 20060101
A61K039/21; A61K 39/12 20060101 A61K039/12; A61K 39/275 20060101
A61K039/275; A61K 39/245 20060101 A61K039/245; A61K 39/235 20060101
A61K039/235 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2004 |
JP |
2004-108599 |
Apr 30, 2004 |
JP |
2004-136756 |
Feb 21, 2005 |
JP |
2005-044639 |
Claims
1-68. (canceled)
69. A method for enhancing an immune response in an animal,
comprising administering to said animal an effective amount of a
viral envelope.
70. The method of claim 69, wherein said immune response is an
antitumor immunity.
71. The method of claim 69, wherein said virus is a species
selected from hemagglutinating virus of Japan, retrovirus,
adenovirus, adeno-associated virus, herpes virus, vaccinia virus,
pox virus and influenza virus.
72. The method of claim 69, wherein said virus is hemagglutinating
virus of Japan.
73. A pharmaceutical composition comprising a cancer
chemotherapeutic encapsulated in a viral envelope vector having an
adjuvanticity.
74. The pharmaceutical composition of claim 73, wherein said cancer
chemotherapeutic is one kind selected from bleomycins,
anthraquinone series carcinostatics, mitomycins, actinomycins,
camptothecins, cisplatins, streptozotocin, 5-fluorouracil (5-FU)
and derivatives thereof, pirarubicin and pharmacologically
acceptable salts thereof.
75. The pharmaceutical composition of claim 74, wherein said
bleomycins include bleomycin and pharmacologically acceptable salts
thereof, and peplomycin and pharmacologically acceptable salts
thereof.
76. The pharmaceutical composition of claim 75, wherein said
bleomycins include bleomycin hydrochloride, bleomycin sulfate and
peplomycin sulfate.
77. The pharmaceutical composition of claim 73, wherein said virus
is a species selected from hemagglutinating virus of Japan,
retrovirus, adenovirus, adeno-associated virus, herpes virus,
vaccinia virus, pox virus and influenza virus.
78. The pharmaceutical composition of claim 73, wherein said cancer
chemotherapeutic is one kind selected from bleomycin hydrochloride,
bleomycin sulfate and peplomycin salfate, and wherein said virus is
hemagglutinating virus of Japan.
79. The pharmaceutical composition of claim 73, which is an
injection.
80. The pharmaceutical composition of claim 73, which is an agent
for the treatment of solid cancer.
81. The pharmaceutical composition of claim 80, wherein said solid
cancer is selected from lung cancer, breast cancer, digestive
cancer, head and neck cancer, gynecologic cancer, urological
cancer, osteochondrosarcoma, malignant lymphoma and cancer unknown
primary.
82. The pharmaceutical composition of claim 81, wherein said
digestive cancer is selected from stomach cancer, colon cancer and
esophagus cancer.
83. The pharmaceutical composition of claim 81, wherein said
urological cancer is selected from prostate cancer, bladder cancer,
kidney cancer, renal pelvic and ureter cancer, testicular tumor,
adrenal tumor and penis cancer.
84. A pharmaceutical composition for the treatment of a urological
cancer, which comprises a hemagglutinating virus of Japan envelope
and an anticancer drug.
85. The pharmaceutical composition of claim 84, wherein said
urological cancer is one kind selected from prostate cancer,
bladder cancer, kidney cancer, renal pelvic and ureter cancer,
testicular tumor, adrenal tumor and penis cancer.
86. The pharmaceutical composition of claim 84, wherein said
anticancer drug is at least one kind selected from adriamycin,
daunomycin, aclarubicin, amrubicin, idarubicin, epirubicin,
pirarubicin, dacarbazine and mitoxantrone.
87. The pharmaceutical composition of claim 84, wherein said
urologic cancer is bladder cancer, and wherein said anticancer drug
is adriamycin.
88. The pharmaceutical composition of claim 87, which is for
intravesical instillation.
89. A method for the treatment of a urological cancer in an animal,
which comprises administering to said animal effective amounts of a
hemagglutinating virus of Japan envelope and an anticancer
drug.
90. The method of claim 89, wherein said urological cancer is one
kind selected from prostate cancer, bladder cancer, kidney cancer,
renal pelvic and ureter cancer, testicular tumor, adrenal tumor and
penis cancer.
91. The method of claim 89, wherein said anticancer drug is at
least one kind selected from adriamycin, daunomycin, aclarubicin,
amrubicin, idarubicin, epirubicin, pirarubicin, dacarbazine and
mitoxantrone.
Description
TECHNICAL FIELD
[0001] The present invention relates to a vehicle for inducing
antitumor immunity in a living organism. More specifically, the
present invention relates to inducing more potent antitumor
immunity by introducing a chemotherapeutic agent, preferably an
anticancer drug, in combination with, or encapsulated in, a virus,
specifically an inactivated virus, particularly an inactivated
hemagglutinating virus of Japan (hereinafter also referred to as
HVJ), into a solid tumor, and further systemically coadministering
an anticancer drug. The present invention also relates to a
pharmaceutical composition exhibiting antitumor action, which
comprises a viral envelope and an anticancer drug.
BACKGROUND ART
[0002] In current cancer treatment, the cure rate is reportedly
about 50%, and healing is generally often achieved by topical
therapies such as surgical therapy and radiotherapy. In particular,
in the treatment of solid cancers, the relative contribution of
chemotherapy, which is a systemic therapy, to healing when it is
used alone, is very low, and it is common practice to use it in
combination with various other therapies.
[0003] In surgical therapy, all visceral cancers are operable; this
therapy is considered to have already reached completion as a
therapeutic approach, and no further improvement in cure rate is
expected. As for radiotherapy, therapeutic results for the
treatment of responsive visceral organs have become nearly
constant, and no further improvement in cure rate is expected, as
with surgical therapy.
[0004] Therefore, because these therapies are no longer likely to
significantly improve the cancer cure rate from now on, the cancer
cure rate cannot be increased from the current level of 50% to
achieve cancer control unless a better chemotherapy is
developed.
[0005] Anticancer drugs used for chemotherapy are intended to
obtain cell-killing effects on cells of high growth potential such
as cancer cells, and cause major damage to normal cells,
particularly to myelocytes and other cells of high cell growth
potential, resulting in a major burden on the patient. This is
because the anticancer drug is delivered by systemic administration
of an injection, so that the anticancer drug reaches not only
cancer cells but also normal cells and kills normal cells to hamper
the functioning of homeostasis.
[0006] However, at present, the efficacy rate of an anticancer drug
administered alone is reported to be roughly about 30%; although it
is hoped that advances in analytical research in genetic
information of genome will enable the selection of appropriate
anticancer drugs in the future, the currently available anticancer
drug therapy is reported to produce a higher prevalence of
side-effects compared with their efficacy.
[0007] This is because normal cells are damaged by the systemic
administration of the anticancer drug. Hence, provided that a
method is established of introducing an anticancer drug
specifically to cancer tissue, and incorporating the agent into
cancer cells, an ideal anticancer drug delivery system would be
realized. Furthermore, if it is possible to encapsulate an
anticancer drug into a vesicle, a therapy could be established that
act selectively on target organs or cells with little influence on
normal cells (side-effects). Additionally, this is considered to
lead to a re-evaluation of anticancer drugs the development of
which has been discontinued due to strong side-effects.
[0008] Additionally, therapeutic results for malignant tumors have
recently been remarkably improved with the advances in
multidisciplinary treatment centered on chemotherapy. In
particular, in hematopoietic tumors such as leukemia and malignant
lymphoma, healing is well expectable when these therapies are used
in combination with hematopoietic stem cell transplantation and the
like. However, no more than toxic effects of anticancer drugs,
radiotherapy and the like are observed in some cases; there is a
limitation in the eradication of tumor cells. Basic research and
clinical observations have shown that the selective elimination of
tumor cells by immune system is important. The immune system acts
by recognizing proteins (peptides) and non-peptide antigens such as
saccharides and lipids, whether the relevant organ is mucosal or
non-mucosal. Upon entry of pathogen in the body, mainly monocytes
migrate to the entry site and exhibit antigen-nonspecific
protective responses via phagocytosis and the like. Natural
immunity against non-peptides such as saccharides and lipids and
the like is first induced, helping the production of various
factors concerning the elimination of pathogens. Subsequently,
lymphocytes that recognize pathogenic peptides grow and
differentiate; B lymphocytes differentiate into antibody-producing
cells and T lymphocytes differentiate into helper T cells,
cytotoxic T cells and the like, which control the immune system,
thus inducing antigen-specific immune responses, i.e., what is
called acquired immunity. There are two types of acquired immunity:
humoral immunity, in which antibodies play the key role, and
cellular immunity, in which T lymphocytes play the key role. Which
is the prevalent type of immunity, whether cellular immunity or
humoral immunity, depends on which is the prevalent subtype of
helper T cells, whether Th1 or Th2. When the immune state is
inclined to Th1-dominant, cellular immunity will prevail; when the
immune state is inclined to Th2-dominant, humoral immunity will
prevail. The two types of immunity occur in a mutual balance; these
immune states rely on cytokines, which are humoral molecules
secreted by various cells. As Th1 type cytokines, IL-12, IFN.gamma.
and the like can be mentioned; as Th2 type cytokines, IL-4, IL-5
and the like can be mentioned.
[0009] Considering immunity, particularly tumor immunity, it has
been reported that CD8-positive cytotoxic T cells (CTL) and
CD4-positive helper T cells play a very important role (North R J.
1984, Greenberg P D. 1991, Pardoll D M. 1998). In particular, it
has been reported that CD8-positive T cells (CTL) of immunized
animals directly injured target cells in vitro (Wanger H. 1980),
and that tumor resistance was conferred to non-immunized animals by
adoptive immunity (North R J. 1984, Greenberg P D. 1991).
Therefore, how efficiently tumor-specific CTL can be induced is
important in the development of antitumor therapy. In antitumor
immunity with CTL, CTL recognizes a complex of a major
histocompatibility complex (MHC) class I molecule and a tumor
antigen-derived peptide, expressed on the tumor cell surface, via a
T cell receptor and introduces perforin and the like into tumor
cells, thereby exhibiting its cytotoxicity. In the induction of
tumor-specific CTL, a focus is placed on first identifying a target
antigen peptide that can be specifically expressed in tumor cells,
processed in cells, and presented to MHC as a peptide fragment;
many peptide molecules that produce high titers of IgG antibodies
have been found by the serological analysis of recombinant cDNA
expression library (SEREX) method.
[0010] However, it is difficult to describe tumor immunity solely
based on the identification of a tumor peptide. Many challenges
remain unresolved, including how efficiently to present the
identified peptide on the cell surface in vivo, and the expression
of CD80/CD86, which are co-stimulatory molecules. Speaking in
detail, it has been reported that when the peptide is efficiently
expressed, but the expression of CD80/CD86 and the like which are
co-stimulatory molecules, is low, and the antigen signal alone is
transmitted, growth of antigen-specific T cells does not occur and,
what is more, T cell anergy develops in the cells that express the
antigen (Gribben J G. 1996). In leukemia cells, many gene
abnormalities have been shown to be involved in the mechanism for
acquiring the growth dominance; it is considered that a specific
abnormal protein formed due to such a gene abnormality is
expressed, processed and fragmented, and the resulting peptide is
presented to the groove of MHC on the cell surface. However, it has
been reported to be difficult to induce an effective immune
reaction to leukemia (Hirano N. 1996) because the expression of
co-stimulatory molecules such as CD80/CD86 on the surface of many
leukemia cells is insufficient despite the expression of such a
leukemia-specific antigen thereby.
[0011] Also, as a recent finding, it has been reported that the
reason why tumor rejection does not occur despite an increase of
CTL is that the infiltration of immune cells and inflammatory cells
is prevented by stroma cells present in the vicinity of the tumor
(more than 90% of the tumor tissues of breast cancer, pancreatic
cancer, and stomach cancer comprise interstitial fibroblasts), and
that the tumor cell regression effect was dramatically increased by
removing these disturbances (Yu P. 2004); the infiltration of
inflammatory cells and immune cells in tumor cells has been
considered to be an important factor concerning tumor
regression.
[0012] Hence, successful utilization of tumor immunity relies on
how efficiently tumor-specific CTL can be induced, and the
following problems arise: [0013] Whether or not any tumor-specific
antigen has been identified? [0014] What is to do if no
tumor-specific antigen has not been identified? [0015] How
efficiently is an antigen presented to immunity-inducing cells
(dendritic cells and the like)? [0016] How efficiently is the
maturation of immunity-inducing cells (dendritic cells and the
like) achieved? [0017] How to induce tumor immunity (mainly by CTL)
with these points in mind [0018] Efficient infiltration of immune
cells in a tumor tissue
[0019] Of the above-described problems, whether or not tumor
immunity (mainly by CTL) is induced represents the most important
requirement; an adjuvant that induces immunity is required for the
induction of CTL. As an adjuvant capable of shifting the immune
state to Th1-dominant, a patent application for "an adjuvant
composed of HVJ-charged liposome" has been published
(JP2001-302541A). Some reports are available on the effects thereof
as a tumor vaccine [Anticancer Res., (19): 5367-5374. 1999, Ihshda
H et al.; Hum. Gene. Ther., (10): 2719-2724. 1999, Zhou W Z. et
al.; Gene. Ther., (6): 1768-1773. 1999. Zhou W Z. et al.; Mol.
Ther., 5(3), 291-299, 2002. Tanaka M. et al.]. However, concerning
the hemagglutinating virus of Japan envelope not in the form of
liposome (hereinafter also referred to as HVJ-E), no such effects
have been reported to date.
[0020] HVJ-E is a vector constructed on the basis of HVJ
(JP2002-65278A); it permits the inclusion of a plasmid,
oligo-DNA/RNA, protein, peptide, or low molecular compound in a
vector vehicle, permits the introduction of the included sample
into cells in the vicinity of the vector vehicle in vitro and in
vivo, and enables the fusion of cells with each other by the action
of the F protein, which is an envelope protein. The present
inventors investigated tumor immunity using HVJ-E by making use of
the above-described advantages.
[0021] Although immune gene therapy is the most suitable method of
gene therapy for the metastasis suppression or recurrence
prevention of cancer, its effect remains unsatisfactory worldwide.
As a cause for this, it is postulated that tumor immunity cannot be
enhanced to an extent sufficient to achieve successful treatment.
To this end, we have been developing a gene transfer vector and a
method of gene expression. As a result, it has become possible to
administer an inactivated HVJ envelope as the adjuvant and an
anticancer drug, that is encapsulated in the vector, or used in
combination therewith, directly into a solid tumor, with
coadministration of another anticancer drug, thereby inducing
tumor-specific antitumor immunity. In this case, no tumor-specific
antigen peptide is required; in other words, this is applicable to
a broad range of tumors for which no tumor peptides have been
identified. Furthermore, the present invention is groundbreaking in
that the situation wherein CTL cells cannot reach the tumor site
due to stroma cells and the like surrounding solid tumor tissue,
despite induction of CTLs, and hence cannot kill the tumor cells of
the solid tumor, so that no tumor regression is observed, can be
overcome by using HVJ-E and an anticancer drug in combination.
DISCLOSURE OF THE INVENTION
[0022] The present inventors diligently investigated to solve the
above-described problems and succeeded in developing a
pharmaceutical composition comprising a viral envelope vector in
combination with, or encapsulating, a chemotherapeutic agent, as an
active ingredient.
[0023] Furthermore, as a method more likely to find a clinical
application, the inventors introduced an anticancer drug
encapsulated in, or used in combination with, a viral envelope
vector, directly into a tumor, with coadministration of another
anticancer drug, thereby succeeding in inducing tumor cell-specific
antitumor immunity also due to the adjuvant action of HVJ-E to
regress the tumor.
[0024] Therefore, specifically, the present invention provides a
pharmaceutical composition wherein an anticancer drug or the like
is encapsulated in, or used in combination with, for example, an
inactivated HVJ-E vector or the like having the capability of
encapsulating a foreign gene.
[0025] The present invention further provides a method comprising
introducing an HVJ-E vector and the like containing an anticancer
drug into a tumor, in combination with administration of another
anticancer drug, thereby inducing high antitumor immunity also
attributable to the adjuvant effect of the vector to regress the
tumor.
[0026] The present invention also provides a pharmaceutical
composition for treating a solid cancer, particularly urological
cancer, which comprises the hemagglutinating virus of Japan
envelope and an anticancer drug not encapsulated in the
envelope.
[0027] There are four points of the gist of the present
invention.
[0028] First, HVJ is used. Tumor immunity is induced by the
adjuvant effect of HVJ.
[0029] The second gist resides in that efficient tumor immunity was
successfully induced by introducing bleomycin (bleomycin
hydrochloride, Bleo for Injection: Nippon Kayaku Co., Ltd.),
previously encapsulated in an HVJ-E vector, into transplanted tumor
tissue, in combination with systemically administered cisplatin
(CDDP, Randa Injection: Nippon Kayaku Co., Ltd.).
[0030] Regarding the third gist, the induced CTL cells can be
highly efficiently accumulated in the solid tumor.
[0031] The third gist also resides in that the induced tumor
immunity also retains essential immune actions, including
eradication of homologous tumor cells, and that feasibility for use
as what is called a neoadjuvant was demonstrated, including the
elimination of metastatic tumor by the actions, the prevention of
metastasis after surgical operation for surgical elimination of a
solid tumor by conducting the treatment in advance, and the killing
of metastatic tumors in lymph nodes in the vicinity of the primary
tumor to minimize the area to be resected by surgical
treatment.
[0032] Regarding the fourth gist, a significant antitumor effect
was obtained by using HVJ-E and an anticancer drug not encapsulated
in HVJ-E in combination.
[0033] Accordingly, the present invention provides the following:
[0034] (1) An immune adjuvant comprising a viral envelope. [0035]
(2) The adjuvant described in (1) wherein the aforementioned
adjuvant is an adjuvant for enhancing an immune response. [0036]
(3) The adjuvant described in (1) or (2) wherein the aforementioned
adjuvant is an adjuvant for enhancing an antitumor immunity. [0037]
(4) The adjuvant described in (1) to (3) wherein the aforementioned
virus is a virus belonging to a family selected from the group
consisting of Retroviridae, Togaviridae, Coronaviridae,
Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae,
Rhabdoviridae, Poxviridae, Herpesviridae, Baculoviridae, and
Hepadnaviridae. [0038] (5) The adjuvant described in (1) to (4)
wherein the aforementioned virus is a species selected from
hemagglutinating virus of Japan, retrovirus, adenovirus,
adeno-associated virus, herpes virus, vaccinia virus, pox virus and
influenza virus. [0039] (6) The adjuvant described in (1) to (5)
wherein the aforementioned virus is hemagglutinating virus of
Japan. [0040] (7) A viral envelope for use as an immune adjuvant.
[0041] (8) A hemagglutinating virus of Japan envelope for use as an
immune adjuvant. [0042] (9) A hemagglutinating virus of Japan
envelope for use as an antitumor immunity adjuvant. [0043] (10) A
use of a viral envelope and cisplatin for improving the tumor
antigen-presenting capability of antigen-presenting cell, which
results in the accumulation of cytotoxic T-lymphocyte (CTL) cells
in a tumor tissue. [0044] (11) A use of a viral envelope, cisplatin
and a chemotherapeutic agent for improving the tumor
antigen-presenting capability of antigen-presenting cell, which
results in the accumulation of cytotoxic T-lymphocyte cells in a
tumor tissue. [0045] (12) The use described in (10) or (11) wherein
the aforementioned viral envelope is a hemagglutinating virus of
Japan envelope (HVJ-E). [0046] (13) The use described in (10) to
(12) wherein the aforementioned chemotherapeutic agent is
bleomycin. [0047] (14) A method of improving the tumor
antigen-presenting capability of antigen-presenting cell, which
results in the accumulation of cytotoxic T-lymphocyte cells in a
tumor tissue, which method uses a viral envelope and cisplatin.
[0048] (15) A pharmaceutical composition for improving the tumor
antigen-presenting capability of antigen-presenting cell, which
results in the accumulation of cytotoxic T-lymphocyte cells in a
tumor tissue, which composition comprises a viral envelope and
cisplatin. [0049] (16) A use of a viral envelope and cisplatin for
the manufacture of a pharmaceutical for improving the tumor
antigen-presenting capability of antigen-presenting cell, which
results in the accumulation of cytotoxic T-lymphocyte cells in a
tumor tissue. [0050] (17) A pharmaceutical composition comprising a
chemotherapeutic encapsulated in a viral envelope vector having an
adjuvanticity as an active ingredient. [0051] (18) The
pharmaceutical composition described in (17) wherein the
chemotherapeutic is an anticancer drug. [0052] (19) The
pharmaceutical composition described in (17) or (18) wherein the
chemotherapeutic is one or more kinds selected from bleomycins,
anthraquinone series carcinostatics, mitomycins, actinomycins,
camptothecines, cisplatins, streptozotocin, 5-fluorouracil (5-FU)
and derivatives thereof, pirarubicin, and pharmacologically
acceptable salts thereof. [0053] (20) The pharmaceutical
composition described in (17) to (19) wherein the bleomycins are
bleomycin and pharmacologically acceptable salts thereof, and
peplomycin and pharmacologically acceptable salts thereof. [0054]
(21) The pharmaceutical composition described in (17) to (20)
wherein the bleomycins are bleomycin hydrochloride, bleomycin
sulfate and peplomycin sulfate. [0055] (22) The pharmaceutical
composition described in (17) to (21) wherein the virus having an
adjuvanticity is derived from a virus belonging to a family
selected from the group consisting of Retroviridae, Togaviridae,
Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae,
Bunyaviridae, Rhabdoviridae, Poxviridae, Herpesviridae,
Baculoviridae, and Hepadnaviridae. [0056] (23) The pharmaceutical
composition described in (17) to (22) wherein the aforementioned
virus is one kind selected from hemagglutinating virus of Japan,
retrovirus, adenovirus, adeno-associated virus, herpes virus,
vaccinia virus, pox virus and influenza virus. [0057] (24) The
pharmaceutical composition described in (17) to (23) wherein the
chemotherapeutic is one or more kinds selected from bleomycin
hydrochloride, bleomycin sulfate and peplomycin sulfate, and
wherein the virus is hemagglutinating virus of Japan. [0058] (25)
The pharmaceutical composition described in (17) to (24), which is
an injection. [0059] (26) The pharmaceutical composition described
in (17) to (25), which is a therapeutic agent for a solid cancer.
[0060] (27) The pharmaceutical composition described in (26)
wherein the solid cancer is one kind selected from lung cancer,
breast cancer, digestive cancer, head and neck cancer, gynecologic
cancer, urological cancer, osteochondrosarcoma, malignant lymphoma
and cancer unknown primary. [0061] (28) The pharmaceutical
composition described in (27) wherein the digestive cancer is one
kind selected from stomach cancer, colon cancer and esophagus
cancer. [0062] (29) The pharmaceutical composition described in
(27) wherein the head and neck cancer is one kind selected from
maxillary cancer, tongue cancer, lip cancer, pharynx cancer, larynx
cancer and mouth cancer. [0063] (30) The pharmaceutical composition
described in (27) wherein the gynecologic cancer is one kind
selected from uterine cancer, ovarian cancer and uterine cervix
cancer. [0064] (31) The pharmaceutical composition described in
(27) wherein the urological cancer is one kind selected from
prostate cancer, bladder cancer, kidney cancer, renal pelvic and
ureteral cancer, testicular tumor, adrenal tumor and penis cancer.
[0065] (32) A pharmaceutical composition comprising a viral
envelope vector having an adjuvanticity as an active ingredient,
which is subjected to use in combination with a chemotherapeutic.
[0066] (33) The pharmaceutical composition described in (32)
wherein the viral envelope vector having an adjuvanticity and the
chemotherapeutic are contained in a preparation. [0067] (34) A
pharmaceutical composition for inducing antitumor immunity in a
living organism, and treating a solid tumor, which comprises an
anticancer drug or immunostimulant encapsulated in a
hemagglutinating virus of Japan envelope. [0068] (35) A
pharmaceutical composition for inducing antitumor immunity in a
living organism, and treating a solid tumor, which comprises an
anticancer drug or immunostimulant encapsulated in a
hemagglutinating virus of Japan envelope, and an additional
anticancer drug. [0069] (36) The pharmaceutical composition
described in (34) or (35) wherein the hemagglutinating virus of
Japan envelope is not in the form of liposome. [0070] (37) The
pharmaceutical composition described in (34) to (36) wherein the
anticancer drug is bleomycin or a pharmacologically acceptable salt
thereof, or peplomycin or a pharmacologically acceptable salt
thereof. [0071] (38) The pharmaceutical composition described in
(34) or (35) wherein the immunostimulant is a protein comprising
granulocyte-macrophage colony-stimulating factor (GM-CSF). [0072]
(39) A pharmaceutical composition for inducing antitumor immunity
in a living organism, and treating a solid tumor, which comprises a
hemagglutinating virus of Japan envelope as an active ingredient,
and which is subjected to use in combination with an anticancer
drug or immunostimulant. [0073] (40) A pharmaceutical composition
for inducing antitumor immunity in a living organism, and treating
a solid tumor, which comprises a hemagglutinating virus of Japan
envelope as an active ingredient, and which is subjected to use in
combination with an anticancer drug or immunostimulant and an
additional anticancer drug. [0074] (41) A method of inducing
antitumor immunity in a living organism to treat a solid tumor,
which comprises administering a pharmaceutical composition
comprising an anticancer drug or immunostimulant encapsulated in a
hemagglutinating virus of Japan envelope. [0075] (42) A method of
inducing antitumor immunity in a living organism to treat a solid
tumor, which comprises administering a pharmaceutical composition
comprising an additional anticancer drug in addition to an
anticancer drug or immunostimulant encapsulated in hemagglutinating
virus of Japan envelope. [0076] (43) The method described in (41)
or (42) wherein the hemagglutinating virus of Japan envelope is not
in the form of liposome. [0077] (44) The method described in (41)
to (43) wherein the anticancer drug is bleomycin or a
pharmacologically acceptable salt thereof, or peplomycin or a
pharmacologically acceptable salt thereof. [0078] (45) The method
described in (41) to (44) wherein the immunostimulant is a protein
comprising granulocyte-macrophage colony-stimulating factor
(GM-CSF). [0079] (46) A use of an anticancer drug or
immunostimulant encapsulated in a hemagglutinating virus of Japan
envelope for the manufacture of a pharmaceutical that induces
antitumor immunity in a living organism to treat a solid tumor.
[0080] (47) A use of an anticancer drug or immunostimulant
encapsulated in a hemagglutinating virus of Japan envelope and an
additional anticancer drug for the manufacture of a pharmaceutical
for inducing antitumor immunity in a living organism to treat a
solid tumor. [0081] (48) A use of a hemagglutinating virus of Japan
envelope as an adjuvant for the manufacture of a drug composition
for inducing antitumor immunity in a living organism to treat a
solid tumor. [0082] (49) A use of a hemagglutinating virus of Japan
envelope as an adjuvant and a vector encapsulating an anticancer
drug or immunostimulant for the manufacture of a pharmaceutical for
inducing antitumor immunity in a living organism to treat a solid
tumor. [0083] (50) A use of a hemagglutinating virus of Japan
envelope as an adjuvant and a vector delivering an anticancer drug
or immunostimulant for the manufacture of a pharmaceutical for
inducing antitumor immunity in a living organism to treat a solid
tumor. [0084] (51) A use of a hemagglutinating virus of Japan
envelope and an anticancer drug in combination for inducing tumor
immunity in a living organism. [0085] (52) A use of a
hemagglutinating virus of Japan envelope for introducing cytotoxic
T-lymphocyte into solid tumor tissue to induce an antitumor effect.
[0086] (53) A use of a hemagglutinating virus of Japan envelope in
preoperative auxiliary therapy (neoadjuvant therapy) to induce
antitumor immunity in a living organism. [0087] (54) The use
described in (46) to (53) wherein the hemagglutinating virus of
Japan envelope is not liposome. [0088] (55) The use described in
(46) to (54) wherein the anticancer drug is bleomycin or a
pharmacologically acceptable salt thereof, or peplomycin or a
pharmacologically acceptable salt thereof. [0089] (56) The use
described in (46) to (54) wherein the immunostimulant is a protein
comprising granulocyte-macrophage colony-stimulating factor
(GM-CSF). [0090] (57) A pharmaceutical composition which comprises
a hemagglutinating virus of Japan envelope and an anticancer drug
for the treatment of urological cancer. [0091] (58) The
pharmaceutical composition described in (57) wherein the urological
cancer is one kind selected from prostate cancer, bladder cancer,
kidney cancer, renal pelvic and ureteral cancer, testicular tumor,
adrenal tumor and penis cancer. [0092] (59) The pharmaceutical
composition described in (57) or (58) wherein the anticancer drug
is at least one kind selected from adriamycin, daunomycin,
aclarubicin, amrubicin, idarubicin, epirubicin, pirarubicin,
dacarbazine and mitoxantrone. [0093] (60) A pharmaceutical
composition for the treatment of bladder cancer, which comprises a
hemagglutinating virus of Japan envelope and adriamycin. [0094]
(61) A pharmaceutical composition for intravesical injection for
the treatment of bladder cancer, which comprises a hemagglutinating
virus of Japan envelope and adriamycin. [0095] (62) A use of a
hemagglutinating virus of Japan envelope and adriamycin in
combination for the treatment of bladder cancer. [0096] (63) A
method for the treatment of urological cancer, which comprises
administering a hemagglutinating virus of Japan envelope and an
anticancer drug. [0097] (64) The method described in (63) wherein
the urological cancer is one kind selected from prostate cancer,
bladder cancer, kidney cancer, renal pelvic and ureteral cancer,
testicular tumor, adrenal tumor and penis cancer. [0098] (65) The
method described in (63) or (64) wherein the anticancer drug is at
least one kind selected from adriamycin, daunomycin, aclarubicin,
amrubicin, idarubicin, epirubicin, pirarubicin, dacarbazine and
mitoxantrone. [0099] (66) A use of a hemagglutinating virus of
Japan envelope and an anticancer drug for the manufacture of a
pharmaceutical for the treatment of bladder cancer. [0100] (67) The
use described in (66) wherein the anticancer drug is at least one
kind selected from adriamycin, daunomycin, aclarubicin, amrubicin,
idarubicin, epirubicin, pirarubicin, dacarbazine and mitoxantrone.
[0101] (68) A pharmaceutical composition for the treatment of
urological cancer, which comprises a hemagglutinating virus of
Japan envelope as an active ingredient, and which is subjected to
use in combination with an anticancer drug.
BRIEF DESCRIPTION OF THE DRAWINGS
[0102] FIG. 1 is a graph comparing the tumor volumes of individual
groups.
[0103] FIG. 2 is a graph comparing the antitumor effects of single
and multiple dosing of CDDP and HVJ-E/BLM.
[0104] FIG. 3 is a graph comparing tumor immunity-inducing effects
in a study of rechallenge of isogenic tumor cells. Rechallenge was
performed on Day 15.
[0105] FIG. 4 is a graph comparing tumor immunity-inducing effects
in a study of rechallenge of isogenic different tumor cells.
Rechallenge was performed on Day 0.
[0106] FIG. 5 is a graph showing comparison of the antitumor
effects between CDDP and BLM, and CDDP and BLM encapsulated in
HVJ-E.
[0107] FIG. 6 is a graph showing CTL induction by CTL assay.
[0108] FIG. 7 shows HE staining images of individual tumor tissue
sections. These are hematoxylin-eosin (HE) staining images for (1)
control group [upper right], (2) HVJ-E group [lower right], (3)
BLM-alone group [upper left], and (4) HVJ-E/BLM group [lower
left].
[0109] FIG. 8 shows hematoxylin-eosin (HE) staining images for (5)
CDDP-alone group [upper right], (6) CDDP+HVJ-E/BLM 25 single dosing
group [lower right], and (7) CDDP+HVJ-E/BLM multiple dosing group
[upper left].
[0110] FIG. 9 is a graph comparing the CD-4-positive images for
individual groups.
[0111] FIG. 10 is a graph comparing CD-8-positive images for
individual groups.
[0112] FIG. 11 is a graph comparing changes in tumor volume among
different groups of animals in a mouse model inoculated with
bladder cancer cells.
[0113] FIG. 12 is a graph showing the adriamycin sensitivity of
MB49 cells.
[0114] FIG. 13 is photomicrographs showing infiltration of MB49
cells into the bladder (Group 1 and Group 2, magnification rate
.times.400).
[0115] FIG. 14 is photomicrographs showing infiltration of MB49
cells into the bladder (Group 3 and Group 4, magnification rate
.times.400).
[0116] FIG. 15 is a graph comparing the MB49 take rate in the
bladder (cancer incidence rate) among different groups.
[0117] FIG. 16 is a graph showing the antitumor effects of a
combination of CDDP and HVJ-E (egg-derived).
[0118] FIG. 17 is a graph showing the antitumor effects of a
combination of CDDP and HVJ-E (cell-derived).
[0119] FIG. 18a is a graph showing immunity-inducting action for
IL-12.
[0120] FIG. 18b shows immunity-inducting action for IL-6.
[0121] FIG. 18c is a graph comparing immunity-inducting actions for
IL-6 and IL-12.
[0122] FIG. 19 is a graph showing increases and decreases in CD4+
cells.
[0123] FIG. 20 is a graph comparing increases and decreases in
regulatory T cells (CD4+CD25+).
DETAILED DESCRIPTION OF THE INVENTION
[0124] The present invention is hereinafter described in
detail.
[0125] The viral envelope in the present invention is a membrane
left after removing RNA or DNA from a virus, and is normally
utilized for transfection of a gene, polynucleotide,
oligonucleotide, plasmid and the like encapsulated therein.
Although the viral envelope preferably used in the present
invention may be in the form of liposome or not, it is preferably
in a form other than liposome.
[0126] The kind of the virus is not subject to limitation;
specifically, for example, a virus belonging to a family selected
from the group consisting of Retroviridae, Togaviridae,
Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae,
Bunyaviridae, Rhabdoviridae, Poxviridae, Herpesviridae,
Baculoviridae, and Hepadnaviridae can be mentioned.
[0127] More specifically, as examples of the virus relating to the
present invention, hemagglutinating virus of Japan, retrovirus,
adenovirus, adeno-associated virus, herpes virus, vaccinia virus,
pox virus, influenza virus and the like can be mentioned.
[0128] Of these, hemagglutinating virus of Japan (hereinafter also
referred to as HVJ), which is one of the mouse pneumonia viruses,
can be preferably mentioned.
[0129] Specifically, as examples of HVJ, VR-105, VR-907 and the
like can be purchased from the American Type Culture Collection
(ATCC; address: P.O. Box 1549, Manassas, Va. 20108 USA,
TEL[1]-703-365-2700).
[0130] http://www.atcc.org/SearchCatalogs/longview.cfm?view=av,
152376, VR-105&text=Sendai&max=20
[0131] http://www.atcc.org/SearchCatalogs/longview.cfm?view=av,
1375478, VR-907&text=Sendai&max=20
[0132] Viral envelope vectors are described in more detail in, for
example, JP2001-286282A (WO 01/57204), JP2002-065278A, WO 03/014338
and the like, and can specifically be prepared in accordance with,
for example, Example 8 of JP2001-286282A and the like.
[0133] These viral envelopes can be used as immune adjuvants, and
are effective in enhancing immune responses and enhancing antitumor
effects. Also, when these viral envelopes are used with cisplatin,
it is possible to improve the tumor antigen presenting capability
of antigen-presenting cells, and thus accumulate cytotoxic
T-lymphocyte cells in a tumor tissue.
[0134] Major advantages of using a viral envelope are described in
detail below.
[0135] As stated above, of the viral envelopes, a hemagglutinating
virus of Japan envelope (hereinafter also referred to as HVJ-E) is
preferably used; HVJ-E causes decreased cytokine induction to
dendritic cells, which are antigen-presenting cells, compared with
live virus HVJ. In particular, Examples described below have shown
that IL-12, which is a Th1 cytokine, is decreased, and that the
amount of IL-6 induced is reduced to about 1/2 compared with HVJ.
Even in this case, no increase in Th2 cytokines is observed. Hence,
it was found that when using HVJ-E, the amount of cytokines induced
decreases but the dendritic cell maturation potential is
retained.
[0136] In antitumor immunity, although it has been reported that
when the ratio of regulatory T cells increases, the subsequent
antitumor immunity action is suppressed [Casares N. et al., J
Immunol. Dec. 1, 2003; 171(11): 5931-9, Takahashi T. et al., Int
Immunol. December 1998;10 (12): 1969-80], HVJ-E acts to lower the
ratio of regulatory T cell CD4+CD25+ without influencing the
overall ratio of T cells, as shown in an Example below. Because
this action is not influenced by an anticancer drug, the use of
HVJ-E is highly useful in antitumor immunity.
[0137] Such a viral envelope can also be used for preoperative
auxiliary therapy (neoadjuvant therapy) to induce antitumor
immunity in a living organism.
[0138] Furthermore, in addition to cisplatin, a chemotherapeutic
may be used together. As a chemotherapeutic preferably used here,
bleomycin and the like can be mentioned.
[0139] By the viral envelope of the present invention, a method
enabling the convenient and safe delivery of an anticancer drug
that produces a significant side-effects to the cancer part is
provided. Accordingly, the present invention provides a
pharmaceutical composition comprising a chemotherapeutic
encapsulated in, or used in combination with, a viral envelope
having an adjuvanticity, as an active ingredient.
[0140] The chemotherapeutic used in the present invention is not
subject to limitation, as long as it is a low-molecular compound
that acts directly on cells; for example, in Seikagaku Jiten, 3rd
edition, published by Tokyo Kagaku Dojin, it is stated that
"currently, the coverage of targets of a therapy using chemical
substances of highly selective toxicity, i.e., chemotherapy, has
been expanded to cover malignant tumors, as well as microbial
infections"; antimicrobial drugs, anticancer drugs and the like are
of course included in the scope of the chemotherapeutic used in the
present invention.
[0141] The chemotherapeutic in the present invention is preferably
an anticancer drug or an antimicrobial drug. The term anticancer
drug as used herein refers to a concept including what are called
carcinostatics and antitumor drugs; the terms anticancer,
carcinostatics, and antitumor as used herein are synonymous with
each other.
[0142] As specific examples of the anticancer drug, bleomycins,
anthraquinone (anthracycline) series carcinostatics such as
adriamycin (doxorubicin), daunomycin (daunorubicin), aclarubicin,
amrubicin, idarubicin, epirubicin, pirarubicin, and mitoxantrone,
mitomycins, actinomycins, camptothecines such as irinotecan,
cisplatins, streptozotocin, 5-fluorouracil (5-FU) and derivatives
thereof, pirarubicin, dacarbazine and pharmacologically acceptable
salts thereof can be mentioned.
[0143] Of these chemotherapeutics, preference is given to
anticancer drugs; more preferably, bleomycins, cisplatins or
adriamycin can be mentioned; specifically bleomycin or a
pharmacologically acceptable salt thereof, or peplomycin or a
pharmacologically acceptable salt thereof can be mentioned; more
specifically bleomycin hydrochloride, bleomycin sulfate, and
peplomycin sulfate can be mentioned.
[0144] Cisplatins in the present invention specifically mean
carcinostatic platinum complexes such as cisplatin (CDDP),
carboplatin [paraplatin] (CBDCA), and nedaplatin.
[0145] As specific examples of the antimicrobial drug,
oxophosphoric acid, ormetoprim (OMP), trimethoprim, sulfonamids,
phosphomycin, penicillin-series antimicrobial drugs,
cephalosporin-series antimicrobial drugs, vancomycin,
tetracycline-series antimicrobial drugs, rifampicin,
fluoroquinone-series antimicrobial drugs and the like can be
mentioned.
[0146] When using a pharmaceutical composition of the present
invention as an anticancer agent, the kind of cancer for which the
pharmaceutical composition is indicated is not subject to
limitation; specifically, a solid cancer, blood cell cancer and the
like can be mentioned. Of these, a solid cancer is a suitable
target.
[0147] More specifically, as examples of the solid cancer, lung
cancer, breast cancer, digestive cancer, head and neck cancer,
gynecologic cancer, urological cancer, osteochondrosarcoma,
malignant lymphoma, a cancer unknown primary, skin cancer, skin
malignant tumor, glioma, thyroid cancer and the like can be
mentioned; still more preferably, as examples of the digestive
cancer, stomach cancer, colon cancer, esophagus cancer and the like
can be mentioned; as the head and neck cancer, maxillary cancer,
tongue cancer, lip cancer, pharynx cancer, larynx cancer, mouth
cancer and the like can be mentioned; as the gynecological cancer,
uterine cancer, ovarian cancer, uterine cervix cancer and the like
can be mentioned; as the urological cancer, prostate cancer,
bladder cancer, kidney cancer, renal pelvic and ureteral cancer,
testicular tumor, adrenal tumor, penis cancer and the like can be
mentioned.
[0148] Of these solid cancers, skin cancer, skin malignant tumors,
head and neck cancers (maxillary cancer, tongue cancer, lip cancer,
pharynx cancer, mouth cancer and the like), lung cancer
(particularly primary and metastatic squamous cell carcinoma),
esophagus cancer, malignant lymphoma (reticulosarcoma,
lymphosarcoma, Hodgkin's disease and the like), uterine cervix
cancer, glioma, thyroid cancer, prostate cancer, and bladder cancer
can be mentioned as more suitable targets.
[0149] An immunostimulant may be further encapsulated in, or used
in combination with the viral envelope contained in the
above-described pharmaceutical composition. Here, as a preferable
immunostimulant, proteins, including granulocyte-macrophage
colony-stimulating factor (GM-CSF), and the like can be
mentioned.
[0150] The pharmaceutical composition of the present invention,
which comprises an anticancer drug or immunostimulant, is capable
of inducing antitumor immunity in a living organism to treat a
solid tumor. As used herein, "treat" refers to an action to treat
or suppress a solid tumor, specifically means that the solid tumor
is regressed, eradicated and the like by injecting the
pharmaceutical composition directly into an affected portion or
systemically administering the same.
[0151] In the step for encapsulating a chemotherapeutic or
immunostimulant in a viral envelope, it is preferable to use a
surfactant; as specific examples of the surfactant, Triton X100,
deoxycholic acid or a salt thereof, cholic acid or a salt thereof,
dodecylmaltoside and the like can be mentioned. As the salt of
deoxycholic acid, sodium deoxycholate is preferable; as the salt of
cholic acid, sodium cholate is preferable.
[0152] Specifically, in the case of an inactivated hemagglutinating
virus of Japan envelope (HVJ-E) in which an anticancer drug or
immunostimulant is to be encapsulated, the anticancer drug or
immunostimulant is dissolved in buffer solution. The buffer
solution used here is not subject to limitation; specifically, for
example, TE buffer solution (10 mM Tris, 1 mM EDTA [pH 8.0]), PBS
(phosphate buffer solution) and the like can be chosen and used as
appropriate, with preference given to a buffer solution having a pH
of 6 to 9.
[0153] In addition to the anticancer drug or immunostimulant
encapsulated in the viral envelope, the above-described
pharmaceutical composition may comprise an additional anticancer
drug. As specific examples of the additional anticancer drug, those
mentioned to exemplify the aforementioned anticancer drug and the
like can be mentioned.
[0154] Regarding the method of administration and dosage form of
the above-described pharmaceutical composition, any of oral
administration and non-oral administration is acceptable; as
preparations for oral administration, solid preparations such as
powders, granules, capsules, tablets, and chewable tables, and
liquid preparations such as solutions and syrups, can be mentioned;
as preparations for non-oral administration, injections, ointments,
sprays and the like can be mentioned. Preferably, the
above-described pharmaceutical composition is given as a
preparation for non-oral administration, more preferably an
injection.
[0155] As the non-human animal, laboratory animals, as well as
domestic animals and poultry, are included. The method of
administration to a non-human animal may be addition to feed.
[0156] The dosage of the above-described pharmaceutical composition
varies depending on recipient patient age, body weight, and
pathologic condition, method of administration and the like; in a
viral envelope composition encapsulating an anticancer drug, it is
preferable to administer a titer of normally 15 to 30 mg in the
case of, for example, bleomycin, or administer normally about 10 to
100 mg/m.sup.2 in the case of cisplatin, based on daily dosage per
adult. In a viral envelope composition including an
immunostimulant, it is preferable to administer normally about
1,000,000 to 20,000,000 units in the case of, for example,
GM-CSF.
[0157] Also, based on HVJ-E, normally 40 to 400,000 HAU, preferably
1,200 to 120,000 HAU, and more preferably 4,000 to 40,000 HAU is
administered.
[0158] In the case of a pharmaceutical composition comprising a
viral envelope encapsulating, or used in combination with, an
anticancer drug, and an additional anticancer drug, the HVJ-E
encapsulating, or used in combination with, the anticancer drug,
and the additional anticancer drug, may be prepared separately or
together, or may be prepared as a preparation comprising all of
them. If they are administered as separate preparations, the routes
of administration and dosage forms thereof may be the same or
different, and the timings of administration thereof may be the
same or different. These are determined as appropriate according to
the kinds and effects of the anticancer drugs used in
combination.
[0159] "In combination" as used herein is understood to mean that
the timings of administration thereof may be the same or different,
and are not subject to limitation.
[0160] The above-described pharmaceutical composition can be
prepared as solid preparations such as powders, granules, capsules,
tablets, and chewable tables, liquid preparations such as solutions
and syrups, injections, sprays, ointments and the like by ordinary
methods.
[0161] As required in pharmaceutical making, an appropriate
pharmaceutically acceptable carrier, for example, an excipient, a
binder, a lubricant, a solvent, a disintegrant, a solubilizer, a
suspending agent, an emulsifier, an isotonizing agent, a
stabilizer, a soothing agent, an antiseptic, an antioxidant, a
corrective, a coloring agent and the like are formulated.
[0162] As the excipient, organic excipients such as saccharides
such as lactose, glucose, and D-mannitol, starches, and celluloses
such as crystalline cellulose, inorganic excipients such as calcium
carbonate and kaolin and the like can be mentioned; as the binder,
gelatinized starch, gelatin, gum arabic, methylcellulose,
carboxymethylcellulose, carboxymethylcellulose sodium, crystalline
cellulose, D-mannitol, trehalose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl
alcohol and the like can be mentioned; as the lubricant, stearic
acid, fatty acid salts such as stearates, talc, silicates and the
like can be mentioned; as the solvent, purified water,
physiological saline and the like can be mentioned; as the
disintegrant, low-substitution hydroxypropylcellulose, chemically
modified cellulose and starches and the like can be mentioned; as
the solubilizer, polyethylene glycol, propylene glycol, trehalose,
benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium
salicylate, sodium acetate and the like can be mentioned; as the
suspending agent or emulsifier, sodium lauryl sulfate, gum arabic,
gelatin, lecithin, monostearic glycerol, polyvinyl alcohol,
polyvinylpyrrolidone, celluloses such as carboxymethylcellulose
sodium, polysorbates, polyoxyethylene hardened castor oil and the
like can be mentioned; as the isotonizing agent, sodium chloride,
potassium chloride, saccharides, glycerin, urea and the like can be
mentioned; as the stabilizer, polyethylene glycol, sodium dextran
sulfate, other amino acids and the like can be mentioned; as the
soothing agent, glucose, calcium gluconate, procaine hydrochloride
and the like can be mentioned; as the antiseptic, paraoxybenzoates,
chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic
acid, sorbic acid and the like can be mentioned; as the
antioxidant, sulfites, ascorbic acid and the like can be mentioned;
as the corrective, sweeteners, flavoring agents and the like in
common use in the pharmaceutical area can be mentioned; as the
coloring agent, coloring agents in common use in the pharmaceutical
area can be mentioned.
[0163] Featuring the present invention, it is also possible to
encapsulate an anticancer drug that causes a severe side-effect or
high toxicity in a viral envelope vector, and deliver the
anticancer drug directly to cells, without leakage of the
anticancer drug into the culture broth in in vitro experiments.
[0164] In in vivo animal experiments, not systemic administration,
but topical administration, of an anticancer drug can be performed,
which enables efficient delivery of the anticancer drug exclusively
to the cancer cells of a solid cancer.
[0165] Furthermore, in human treatment, a viral envelope vector
encapsulating, or used in combination with, an anticancer drug, not
only can be administered alone for chemotherapy, but also can be
topically administered to an advanced cancer patient not permitting
the administration of the anticancer drug to achieve cancer
regression; furthermore, radiotherapy and surgical treatment can be
used in combination to obtain a still better anti-cancer
effect.
[0166] For example, for bladder cancer, topical administration such
as intravesical infusion is also possible.
[0167] A viral envelope vector encapsulating, or used in
combination with, an anticancer drug is transfected into host cells
in in vitro experiments. The procedures to follow in this operation
may employ, for example, a method such as adding a solution of the
viral envelope vector encapsulating, or used in combination with,
an anticancer drug, to the medium for cultured cells.
[0168] For the transfection, provided that the reaction is
performed at 37.degree. C., the reaction time is not less than 30
minutes and up to about 48 hours. An evaluation of the effect is
preferably performed by viable cell counting or WST assay (a method
of counting viable cells: cell counting kit-8, Dojindo
Laboratories).
[0169] Regarding recipients in in vivo animal experiments, in the
case of mouse, for example, it is preferable to use normal mice
that are not immunodeficient mice in the case of transplantation of
isogenic cancer cells, or nude mice or SCID mice in the case of
xenogeneic transplantation.
[0170] It is possible to employ a method such as one comprising
intradermally transplanting cancer cells cultured in a Petri dish
to mice, administering a viral envelope vector encapsulating, or
used in combination with, an anticancer drug, into the grown solid
cancer lesion after growth of the transplanted cells, measuring the
major diameter and minor diameter of the cancer lesion, and
determining the anticancer effect thereof.
[0171] The present invention further provides a pharmaceutical
composition comprising HVJ-E and an anticancer drug. In this case,
the above-described treatment of encapsulating an anticancer drug
into a viral envelope need not be performed.
[0172] As the anticancer drug contained in such a pharmaceutical
composition, bleomycins, anthraquinone (anthracycline) series
carcinostatics such as adriamycin (doxorubicin), daunomycin
(daunorubicin), aclarubicin, amrubicin, idarubicin, epirubicin,
pirarubicin, and mitoxantrone, mitomycins, actinomycins,
camptothecines such as irinotecan, cisplatins, streptozotocin,
5-fluorouracil (5-FU) and derivatives thereof, pirarubicin,
dacarbazine and the like can be mentioned, with preference given to
a bleomycin, a cisplatin or adriamycin.
[0173] When a pharmaceutical composition relating to the present
invention is used as an anticancer agent, the kind of cancer for
which the pharmaceutical composition is indicated is not subject to
limitation; specifically, a solid cancer, blood cell cancer and the
like can be mentioned. Of these, a solid cancer is a suitable
target.
[0174] More specifically, suitably treated solid cancers are, for
example, melanoma, a digestive cancer such as colon cancer, breast
cancer, lung cancer, and urological cancer. More specifically, as
the urological cancer, prostate cancer, bladder cancer, kidney
cancer, renal pelvic and ureteral cancer, testicular tumor, adrenal
tumor or penis cancer and the like can be mentioned.
[0175] The above-described pharmaceutical composition comprises
HVJ-E and an anticancer drug, each of which may be prepared
separately or together, or may be prepared as a preparation
comprising all of them. If they are administered as separate
preparations, the routes of administration and dosage forms thereof
may be the same or different, and the timings of administration
thereof may be the same or different. These are determined as
appropriate according to the kind and effect of the anticancer drug
used in combination.
[0176] Regarding the methods of administration and dosage forms of
the above-described pharmaceutical composition, the same methods of
administration and dosage forms as those for the above-described
pharmaceutical composition and the like can be mentioned, with
preference given to an injection.
[0177] As the non-human animal, laboratory animals, as well as
domestic animals and poultry, are included. The method of
administration to a non-human animal may be addition to feed.
[0178] The dosage of the above-described pharmaceutical composition
varies depending on recipient patient age, body weight, and
pathologic condition, method of administration and the like; for
example, in the case of a pharmaceutical composition comprising
adriamycin and HVJ-E, normally 40 to 400,000 HAU of HVJ-E and 1 to
500 mg of adriamycin, preferably 4,000 to 40,000 HAU of HVJ-E and
about 30 to 60 mg of adriamycin, are administered, based on daily
dosage per adult.
[0179] The above-described pharmaceutical composition can be
prepared as a dosage form by an ordinary method as described
above.
EXAMPLES
[0180] Hereinafter, specific examples are given below to
demonstrate the excellent effects of a pharmaceutical composition
comprising a chemotherapeutic encapsulated in, or used in
combination with, a viral envelope vector having adjuvanticity
according to the present invention, as an active ingredient,
particularly the excellent adjuvant effects of viral envelope
glycoprotein and the like of the viral envelope vector, but the
present invention is never limited by these examples.
Example 1
(1) Study Design
[0181] CT-26 cells derived from mouse colon cancer
(5.times.10.sup.6 cells) were intradermally transplanted to the
backs of 8-week-old male BALB/cAnNCrj mice to yield cancer-bearing
mice. Five days after the transplantation, 0.2 mg/body Platocin
Injection (cisplatin, CDDP) was intraperitoneally administered to
animals wherein the tumor diameter (major diameter) had reached
about 5 mm. Following this administration, HVJ-E containing or not
containing an anticancer drug (bleomycin hydrochloride [BLM]:
Nippon Kayaku Co., Ltd.) and the like were administered into the
tumor in a single dose (on the day after administration of
cisplatin) or in multiple doses (3 times: 1, 5, and 8 days after
administration of cisplatin). Tumor diameters, survival curves, and
immune reactions in the tumor tissue were evaluated.
[0182] Furthermore, CT-26 cells or isogenic mouse-derived Meth-A
cells were transplanted to cancer-bearing mouse receiving the
above-described treatment, and the dynamics of the transplanted
cells were examined. [0183] (Study 1) The groups described below
were established, and the antitumor effects of treatment for
transplanted tumors was evaluated.
[0184] For modeling, (1) a control group, (2) an HVJ-E group, (3) a
BLM-alone group, (4) an HVJ-E/BLM group, (5) a CDDP-alone group,
and (6) a CDDP HVJ-E/BLM single dosing group were established;
tumor volumes were measured 21 days after each treatment, and the
influence on the antitumor effect was examined.
[0185] Group composition, dosages and the like are described in
detail below.
TABLE-US-00001 Test substance Test substance intraperitoneal
intratumoral administration administration group (mg/body)*
(.mu.g/tumor)** Control group physiological saline 0 physiological
saline 0 HVJ-E group physiological saline 0 HVJ-E 0 6.5 .mu.g/tumor
BLM physiological saline 0 BLM 6.5 group 6.5 .mu.g/tumor HVJ-
physiological saline 0 HVJ-E/BLM 6.5 E/BLM group 0.2 mg/body CDDP
CDDP 0.2 physiological saline 0 group 0.2 mg/body CDDP 0.2
HVJ-E/BLM 6.5 CDDP-6.5 .mu.g/ tumor HVJ-E/BLM group *As cisplatin
(CDDP) **As bleomycin (BLM)
[0186] (Study 2) In an attempt to further enhance the antitumor
effect of the (single) treatment by single dosing of CDDP HVJ-E/BLM
given in Study 1, HVJ-E/BLM was administered in multiple doses (3
times) (Study 2-1). Furthermore, to estimate the kind of any
induced antitumor immunity if tumor regression is caused by the
induced immunity in the study, tumor cells [CT26 cells (Study 2-2)
and BALB/c mouse sarcoma cells Meth-A cells (Study 2-3)] were
rechallenged, and the take of the transplanted cells and the
increase or decrease in the tumor volume were examined.
(Study 2-1)
[0187] Tumor volumes were examined 21 days after treatment for (1)
a control group, (2) a CDDP HVJ-E/BLM single dosing group (single
treatment), and (3) a CDDP HVJ-E/BLM 3 dosing group.
TABLE-US-00002 intraperitoneal intratumoral administration
administration group (mg/body)* (.mu.g/tumor)** Control group
physiological physiological saline 0 saline 0 0.2 mg/body CDDP-6.5
.mu.g/ CDDP 0.2 HVJ-E/BLM 6.5 tumor HVJ-E/BLM dosing group 0.2
mg/body CDDP-6.5 .mu.g/ CDDP 0.2 HVJ-E/BLM 6.5 .times. 3 tumor
HVJ-E/BLM 3 dosing group *As cisplatin (CDDP) **As bleomycin
(BLM)
(Study 2-2)
[0188] Prior to re-transplantation of cells, the same procedures as
the above-described study were performed; 15 days after
administration of CDDP, CT-26 cells and Meth-A cells
(5.times.10.sup.6 cells) were separately intradermally transplanted
to a site adjoining the site of the first transplantation of tumor
cells, and rejection of the re-transplanted cells was examined.
TABLE-US-00003 Rechallenged Rechallenged group cells CT-26 cells
MethA Control group 1 (with primary .largecircle. .largecircle.
CT26 administration) Control group 2 (without .largecircle. primary
CT26 administration) primary 0.2 mg/body CDDP-6.5 .mu.g/
.largecircle. tumor HVJ-E/BLM dosing group primary 0.2 mg/body
CDDP-6.5 .mu.g/ .largecircle. .largecircle. tumor HVJ-E/BLM 3
dosing group
[0189] (Study 3) As a supplementary study to the results of Study
2, BLM in an amount equivalent to the amount encapsulated was
administered directly into the tumor, and its effect was
evaluated.
TABLE-US-00004 [0189] intraperitoneal intratumoral administration
administration group (mg/body)* (.mu.g/tumor)** 0.2 mg/body
CDDP-6.5 .mu.g/ CDDP 0.2 HVJ-E/BLM 6.5 .times. 3 tumor HVJ-E/BLM 3
dosing group 0.2 mg/body CDDP-6.5 .mu.g/ CDDP 0.2 6.5 .times. 3
tumor BLM alone 3 dosing group *As cisplatin (CDDP) **As bleomycin
(BLM)
(Study 4) CTL Assay
[0190] To determine whether or not the antitumor immunity effect
observed in the previous study was due to the induction of CT26
cell-specific antitumor immunity, splenocytes recovered from
animals were stimulated with CT26 cells, and a CTL assay using
radiolabeled chromium was performed to determine whether or not
CT26-specific antitumor immunity had been induced.
[0191] In this study, (1) a CT-26 cell non-inoculation group, (2) a
CT-26 cell-alone inoculation group, (3) a CT-26 cell
inoculation+CDDP administration group, and (4) a CT-26 cell
inoculation+CDDP+HVJ-E/BLM 3 dosing group were established and
tested. [0192] (Study 5) To evaluate the infiltration of CD-4,
CD-8, neutrophils, macrophage and the like in the tumor tissues
examined in the study of the antitumor effect of the antitumor
immunity, the following test groups were established, and tumor
tissues were extirpated 9 days after administration of CDDP and
subjected to immunohistological staining.
TABLE-US-00005 [0192] Test substance Test substance intraperitoneal
intratumoral administration administration group (mg/body)*
(.mu.g/tumor)** Control group physiological saline 0 physiological
saline 0 HVJ-E group physiological saline 0 HVJ-E 0 6.5 .mu.g/tumor
BLM physiological saline 0 BLM 6.5 alone dosing group 6.5
.mu.g/tumor HVJ- physiological saline 0 HVJ-E/BLM 6.5 E/BLM dosing
group 0.2 mg/body CDDP CDDP 0.2 physiological saline 0 dosing group
0.2 mg/body CDDP 0.2 HVJ-E/BLM 6.5 CDDP-6.5 .mu.g/ tumor HVJ-E/BLM
dosing group 0.2 mg/body CDDP 0.2 HVJ-E/BLM 6.5 .times. 3 CDDP-6.5
.mu.g/ tumor HVJ-E/BLM 3 dosing group
(2) Experimental Methods
2-1) Cultivation of Tumor Cells
[0193] CT-26 cells derived from BALB/c mouse colon cancer were
cultured using a DMEM medium containing 10% FBS at 37.degree. C. in
the presence of 5% CO.sub.2.
[0194] Cell culture was performed using a 75-cm.sup.2 flask. After
about 80% confluency was reached, passage culture was performed.
After the DMEM (containing 10% FBS) broth was removed, the cells
were washed with 10 mL of phosphate-buffered saline (PBS), 1 mL of
a PBS containing 0.25% trypsin and 1 mmol/L EDTA-2Na was added, and
the cells were detached at 37.degree. C. After 9 mL of DMEM medium
was added, the cells were harvested, and the cells were recovered
via centrifugation (1000 rpm, 5 minutes). After the supernatant was
removed, the cells were diluted with a DMEM medium containing 10%
FBS and cultured.
[0195] BALB/c mouse Meth-A cells were cultured using a DMEM medium
containing 10% FBS at 37.degree. C. in the presence of 5%
CO.sub.2.
2-2) Preparation of Tumor Cell Suspension
[0196] After the culture broth of cells reaching about 80%
confluency was removed, the culture flask was washed with PBS. A
small amount of a PBS containing 0.25% trypsin and 1 mmol/L
EDTA-2Na was added, and the flask was allowed to stand at
37.degree. C. until the cells began detached. The cells were
harvested using DMEM medium and centrifuged (1000 rpm, 5 minutes).
After the supernatant was removed, the cells were suspended in PBS.
After centrifugation was performed again (1000 rpm, 5 minutes) and
the supernatant was removed, the cells were prepared to
5.times.10.sup.7 cells/mL using PBS.
2-3) Mouse Habituation
[0197] During the 16-day period of quarantine and habituation, the
animals had free access to solid food and drinking water.
2-4) Inoculation of Tumor Cells
[0198] After completion of the quarantine and habituation, animals
were shaven with clippers. The tumor cell suspension at 100
.mu.L/site (5.times.10.sup.6 cells/body) was administered
intradermally to the backs of 59 mice using a disposable syringe
and injection needle (26G). On the day following the
administration, the same administration was performed on 57 animals
(non-recipient animals).
2-5) Animal Grouping
[0199] Tumor diameters (major diameter, minor diameter) were
measured 5 days after transplantation (not measured after
grouping). Five days later, animals having a tumor diameter (major
diameter) of about 5 mm were grouped by stratified randomization to
obtain a nearly uniform distribution of mean tumor diameters (major
diameters).
2-6) Administration
[0200] For the CDDP administration group, a single-dose
intraperitoneal administration (1000 .mu.l) was performed using a
disposable injection syringe and injection needle. In the sample
intratumoral administration group, a desired sample was
administered into the tumor (100 .mu.l) after the day of
administration of CDDP.
2-7) Measurement of Tumor Diameters
[0201] The day of administration was taken as Day 0 after
administration. On Days 3, 6, 9, 12, 15, 18, and 21 after
administration, all animals had their tumor diameters measured, and
tumor volume (major diameter.times.minor diameter.times.minor
diameter/2) was calculated.
[0202] Regarding rechallenges of CT-26 cells and Meth-A cells as
well, tumor diameters were measured after each rechallenge.
2-8) CTL Assay
[0203] The spleen was extirpated from each anesthetized animal and
recovered in a Petri dish (6 cm in diameter) containing 3 ml of
RPMI solution. The spleen tissue was squashed between the frosted
portions of two sterile glass slides, splenocytes were separated
from the coat to the maximum possible extent, and the debris was
removed through meshes. After 7 ml of PRMI solution was added, the
cells were centrifuged at 1200 rpm for 10 minutes, and the
supernatant was removed. The cells were washed by the addition of
additional 10 ml of RPMI solution. The washing procedure was
performed again, and the cells obtained by this procedure were
suspended in 5 ml of GIT (containing 10% FCS and antibiotics)
solution. The cells obtained from this operation were counted.
After adjustment to 5.times.10.sup.6 cells/ml, these cells were
sown to a 12-well plate at 2 ml/well. CT-26 cells as stimulating
cells were added to the wells. The stimulating cells were prepared
by adding 100 .mu.l of mitomycin C to a GIT solution containing the
stimulating cells adjusted to 1.times.10.sup.7 cells/ml, and
treating at 37.degree. C. for 1 hour. After the treatment, 9 ml of
GIT solution was added, the cells were centrifuged at 1000 rpm for
5 minutes, and the supernatant was removed, after which the cells
were washed by the addition of 10 ml of GIT solution, the washing
procedure was performed again, and finally the cells were suspended
in 1 ml of GIT solution to yield a cell suspension. The suspension
was adjusted to a cell density of 2.5.times.10.sup.5 cells/ml by
the addition of GIT solution, and 2 ml of the solution was added to
a suspension of the cells recovered from the aforementioned spleen
tissue.
[0204] For the sample without the stimulating cells, 2 ml of GIT
solution alone was added. The sample was cultured for 6 days. In
CTL assay, these cells were used as the target.
[0205] CTL assay was performed using a cultured sample of
splenocytes recovered from the tissue. First, CT-26 cells for use
as target cells were prepared as described below. A solution of
CT-26 cells was adjusted to 1.times.10.sup.7 cells/ml (in GIT
medium), 150 .mu.l of this solution was taken, 150 .mu.l of a Cr
solution, previously radiolabeled at 1 mCi/ml, was added to the
solution, and the cells were cultured at 37.degree. C. for 1 hour.
After 10 ml of GIT solution was added, the cells were washed and
centrifuged at 1000 rpm for 5 minutes, and the cells were
recovered. The supernatant was removed, and the cells were washed
by the addition of 10 ml of GIT solution. This washing procedure
was performed three times in total. The cells obtained by the
operation were suspended in 150 .mu.l of GIT solution, the
suspension was diluted 100 fold to obtain a final concentration of
1.times.10.sup.5 cells/ml, and this diluted suspension was used as
target cells (T) in the procedures described below.
[0206] Effector cells (E) were prepared from treated cells
recovered from the four groups described in the Method section, and
used.
[0207] Actual CTL assay was performed with the ratio of effector
cells (E) and target cells (T), E/T ratio, set at 80, 35 40, 20,
10, and 5. In this operation, 100 .mu.l of target cells
(1.times.10.sup.5 cells/ml) were added to make a total quantity of
200 .mu.l. The sample was treated at 37.degree. C. for 4 hours.
[0208] After completion of the cultivation, the cells and the
debris were removed via centrifugation, 100 .mu.l of the
supernatant was recovered and placed in a .gamma.-counter, and
radioactivity was counted. % specific Cr release was calculated
using the equation below.
% specific Cr release=b-c/(a-c)*100(%) [0209] a: maximum release
(cpm) [0210] 10 b: experimental release (cpm) [0211] c: spontaneous
release (cpm)
[0212] 2-9) The tumor tissue was frozen in liquid nitrogen in
preparation for staining the tumor tissue. The tumor tissue was cut
into sections 8 .mu.m in thickness using a cryostat. The tissue
sections were fixed in -20.degree. C. cold acetone for 15 minutes.
After the sections were washed with water, endogenous avidin-biotin
blocking was performed, and the sections were washed with water.
The sample was reacted with normal rabbit serum and 50-fold diluted
primary antibody [anti-mouse CD8a rat antibody (Ly-Z; Pharmingen)
or anti-mouse CD4a rat antibody (L3T4; Pharmingen)] at 4.degree. C.
overnight. After the sample was washed with 7.5 mM Tris buffer
solution (pH 7.5), it was reacted with 300-fold diluted anti-rat
biotin-labeled rabbit Ig (DAKO) for 30 minutes. After the sample
was washed with 9.5 mM Tris buffer solution (pH 7.5), it was
reacted with 100-fold diluted streptoavidin for 30 minutes. After
the sample was washed with 11.5 mM Tris buffer solution (pH 7.5), a
color was developed with Fast Red. After the sample was washed with
water, the nuclei were stained with hematoxylin.
Results
Results of Study 1
[0213] Tumor volumes for (1) the control group, (2) the HVJ-E
group, (3) the BLM-alone group, (4) the HVJ-E/BLM group, (5) the
CDDP-alone group, and (6) the CDDP+HVJ-E/BLM single dosing group
were measured.
[0214] The study was performed on a total of 10 animals for each
group in two separate sessions involving five animals each. A
noteworthy finding in the study was that the transplanted tumor
cells disappeared apparently and remission occurred in 2 of the 10
test animals in (6) the CDDP+HVJ-E/BLM single dosing group. In one
of the two tests, the mean values of the tumor volumes of the five
animals are plotted on a graph. The mean tumor volumes were 1641
mm.sup.3 for (1) the control group, 1386 mm.sup.3 for (2) the HVJ-E
group, 1303 mm.sup.3 for (3) the BLM-alone group, 718 mm.sup.3 for
(4) the HVJ-E/BLM group, 387 mm.sup.3 for (5) the CDDP-alone group,
and 51 mm.sup.3 for (6) the CDDP HVJ-E/BLM single dosing group. The
mean values relative to the mean tumor volume for the control group
as 100% were 82.9%, 80.0%, 43.0%, 23.2%, and 3.1% for (2) the HVJ-E
group, (3) the BLM-alone group, (4) the HVJ-E/BLM group, (5) the
CDDP-alone group, and (6) the CDDP HVJ-E/BLM single dosing group,
respectively (FIG. 1).
Results of Study 2-1
[0215] Models for (1) the control group, (2) the CDDP+HVJ-E/BLM
single dosing group, and (3) the CDDP+HVJ-E/BLM 3 dosing group
(HVJ-E/BLM administered at 1, 5, and 8 days after administration of
CDDP) were prepared, and effects on tumor rejection were examined
in the (2) the CDDP+HVJ-E/BLM single dosing group and (3) the
CDDP+HVJ-E/BLM 3 dosing group.
[0216] First, as shown in the figure, remission was observed in 1
of the 4 mice in the group (2). Remission was observed in 4 of the
5 mice in the group (3). (FIG. 2)
Results of Studies 2-2 and 2-3
[0217] In the study of rechallenge of isogenic tumor cells (CT-26
cells) performed in Study 2-2, remission occurred in 1 of the 4
mice in the CDDP+HVJ-E/BLM single dosing group; this mouse received
re-inoculation of CT-26 cells and rejected the transplanted cells.
However, in the remaining three animals, the primary tumor cells
were not rejected; these animals received re-transplantation of
CT-26 cells and could not reject the cells after
re-transplantation. On the other hand, 4 of the 5 animals in the
CDDP+HVJ-E/BLM 3 dosing group exhibited rejection. From the results
shown above, it was confirmed that tumor immunity had been induced
(FIG. 3). Whether or not the tumor immunity is specific for CT-26
cells was determined by the next study.
[0218] In the tests performed in Study 2-3, Meth-A cells
transplanted to mice achieving remission by CDDP+HVJ-E/BLM 3 dosing
could not be rejected (FIG. 4).
Results of Study 3
[0219] In the studies performed previously, an antitumor effect was
observed with CDDP+HVJ-E/BLM. However, regarding the effect of BLM
in those studies, the influence of the presence or absence of
HVJ-E, i.e., the influence of the encapsulation of BLM in HVJ-E
(including the adjuvant effect of HVJ), had not been evaluated; in
the present study, the effect with coadministration of CDDP was
evaluated. The results are shown in FIG. 5. As is evident from the
figure, the tumor regression effect of the treatment was much
lower, and the tumor could not be regressed, in the study performed
with BLM not encapsulated in HVJ-E, compared with BLM encapsulated
in HVJ-E. It was found that even when BLM was introduced directly
into tumor tissue with coadministration of CDDP, no tumor
regression effect was observed unless BLM was encapsulated in HVJ-E
and introduced into the tumor tissue.
Results of Study 4
[0220] The results of the experiments performed in Studies 1 and 2
demonstrated the following facts when CDDP was administered
systemically and BLM encapsulated in HVJ-E was administered
directly into tumor tissue. [0221] Tumor tissue can be regressed.
[0222] The effect is enhanced when a sample of BLM encapsulated in
HVJ-E is administered more than one time. [0223] Mice achieving
remission wherein transplanted cells were regressed by the
treatment received re-transplantation of the cells and rejected the
re-transplanted cells. [0224] However, when different isogenic
cells were transplanted to the mice achieving remission, the
transplanted cells could not be rejected.
[0225] Thus, it was estimated that immunity specific for the
transplanted cells was induced in the study; to examine direct
induction of specific immunity, it was examined whether or not
immunity specific for CT-26 cells was induced.
[0226] (1) A CT-26 cell non-inoculation group, (2) a CT-26
cell-alone inoculation group, (3) a CT-26 cell inoculation+CDDP
dosing group, and (4) a CT-26 cell inoculation+CDDP+HVJ-E/BLM 3
dosing group were established, and CTL assay was performed to
examine CT-26 cell-specific CTL. The ratio of effector cells (E)
and target cells (T), E/T ratio, was varied, and CTL induction was
quantified by % specific Cr release. As a result, as seen from FIG.
6, when the ET ratio was 80%, the % specific Cr release was 8.1%
for (1) the CT-26 cell non-inoculation group, 8.1% for (2) the
CT-26 cell-alone inoculation group, 12.9% for (3) the CT-26 cell
inoculation+CDDP dosing group, and 33.5% for (4) the CT-26 cell
inoculation+CDDP+HVJ-E/BLM 3 dosing group.
[0227] From the results above, it was found that CT-26
cell-specific CTL induction occurred in (4) the CT-26 cell
inoculation+CDDP+HVJ-E/BLM 3 dosing group.
Results of Study 5
[0228] (1) A control group, (2) an HVJ-E group, (3) a BLM-alone
group, (4) an HVJ-E/BLM group, (5) a CDDP-alone group, (6) a
CDDP+HVJ-E/BLM single dosing group, and (7) a CDDP, HVJ-E/BLM 3
dosing-group were established; tumor tissues were collected from
the animals at 9 days after CDDP administration, and sections of
the tissues were prepared for each group. First, characteristic
pathological findings observed in the tissues with HE staining
(FIGS. 7 and 8) and anti-CD-4 antibody and anti-CD-8 antibody
specific immunohistological staining (data not shown) are
described. (1) In the control group, malignant cell findings
specific for tumor cells, i.e., nuclear division in many cells and
high nuclear density, were observed. At the center of the tumor
tissue, necrotic cell death was observed. The majority of the cells
had a morphology characteristic of sarcoma cells, i.e., vigorous
growth of CT-26 cells was observed. Almost no CD-4 or CD-8 (FIGS. 9
and 10)-positive cells were observed. In (2) the HVJ-E group, the
findings were generally the same as (1) the control group;
neutrophil infiltration was observed in the vicinity of the needle
prick where HVJ-E was injected. Generally similar findings were
obtained from (3) the BLM-alone group and (4) the HVJ-E/BLM
group.
[0229] Compared with these groups, in (5) the CDDP-alone group,
tumor cell necrotic signs were observed over a wide range of the
tumor tissue. It was considered that the intraperitoneally
administered CDDP spread in the tumor tissue via blood vessels and
the like and affected the tumor cells by its effect as an
anticancer drug. However, with administration of CDDP alone, not
all tumor cells were eradicated, with some tumor cells remaining;
it was postulated that the tumor growth observed 9 days after CDDP
administration was attributable to the remaining cells. In (6) the
CDDP HVJ-E/BLM single dosing group, tumor cells decreased
generally, and a considerable number of tumor cells disappeared.
The day this sample was recovered was Day 9 of administration of
CDDP, when a remission state had not been reached; it remains
unknown whether or not this sample can achieve remission. It should
be noted, however, that the numbers of CD-4- and CD-8-positive
cells (FIGS. 9 and 10) increased but were smaller than those
obtained with the multiple dosing of HVJ-E/BLM described below. In
(3) the CDDP HVJ-E/BLM 3 dosing group, no or almost no tumor cells
were observed, antigen fibers showed denaturation and necrosis,
with liquefied sites observed. Generally, there were a small number
of cells, and neutrophil infiltration was observed over a wide
range. Characteristically, anti-CD-4 antibody- and anti-CD-8
antibody-positive cells (FIGS. 9 and 10), which were not
conspicuously in other tissues, were observed over a wide range. It
has been reported that anti-CD-8 antibody positive cells that
infiltrate in a tumor tissue are mostly CTL cells; judging from
this finding, combined with the results of Study 4, it was
postulated that in (3) the tumor remission observed in the CDDP
HVJ-E/BLM 3 dosing group was due to CTL.
[0230] In this Example, it was shown that (1) the vaccine effect of
dendritic cells increased with the addition of CpG-ODN, and that
(2) antitumor immunity was induced by the synergistic effect of
intratumoral administration of HVJ-E/BLM and systemic
administration of CDDP.
[0231] As characteristics of the present invention, the following
can be mentioned. [0232] Tumor remission was observed. [0233] As a
reason for the remission, an effect of infiltration of
CD-8-positive cells in the tumor tissue, i.e., tumor cell-specific
CTL, is likely. [0234] Because efficient tumor immunity cannot be
induced solely by administration of HVJ-E/BLM or CDDP alone, their
combination is important. [0235] When CDDP was administered, its
effect was weak but covered the entire tumor tissue, and subsequent
administration of HVJ-E/BLM efficiently delivered HVJ-E/BLM to the
tumor tissue made brittle by the CDDP, and the HVJ-E/BLM fully
exhibited the effect thereof. [0236] In this process, HVJ-E acts as
an adjuvant to induce tumor immunity.
Captions for the Immunostaining Images
Captions for the HE Staining Images in FIGS. 7 and 8
[0237] In the controls, malignant findings are observed, including
high nuclear density, hyper chromatin, lack of nuclear size
uniformity, and high frequency of nuclear division, in the CT-26
cancer cells. Similar findings of this phenomenon were observed
with the administration of HVJ-E, BLM, and HVJ-E/BLM, though there
was some variation. Neutrophil and macrophage infiltration was
observed in the necrotized portions. In the CDDP administration
samples, vacuolar degeneration is observed, with neutrophils,
macrophage and the like infiltrating in the cancer cells; it is
seen that these cells surround the cancer cells. With single dosing
of CDDP+HVJ-E/BLM, the number of tumor cells decreased, whereas
many lymphatic cells are observed. The HE staining density
decreased, showing more pinkish images. This provides evidence for
a reduction in the nucleic acid concentration in the cells, and is
considered to be due to a reduction in the cell density of the
cancer cells. With three dosing of CDDP+HVJ-E/BLM, almost no cancer
cells were observed, with only a few cells showing cell division,
and the cells lost the capability of growing. Many portions showed
denaturation or necrosis, inflammatory cell infiltration was
observed over a wide range, and many lymphocytic cells were
observed.
Example 2
(1) Study Design
B49 Cell-Inoculated Mouse Model
[0238] This experimental model can be prepared in accordance with,
for example, a method described in Anticancer Res., 2004,
24(4):2225-30, and the like.
[0239] Specifically, 2.times.10.sup.6 MB49 cells were intradermally
inoculated to the backs of B6 mice, and the animals were allowed to
stand for 5 days; a sample for each of the groups G1 to G6 was
inoculated three times to the tumors of animals having tumor
diameters of 7-8 mm, and their effects were evaluated. [0240] G1;
physiological saline (control) [0241] G2; adriamycin (ADM) 20 .mu.g
[0242] G3; ADM 100 .mu.g [0243] G4; ADM 20 .mu.g+HVJ-E 5000 HAU
[0244] G5; ADM 100 .mu.g+HVJ-E 5000 HAU [0245] G6; HVJ-E 5000
HAU
(2) Results
[0246] Changes over time in tumor volume (mm.sup.3) for individual
groups are shown below. (see FIG. 11)
TABLE-US-00006 Group G1 G2 G3 G4 G5 G6 Day 0 120 128.4 119 125.3
117.6 120.5 5 351.1 303.2 285.8 266.6 199.1 423.6 7 442.5 330 291.2
190.6 152.6 734.1 9 717.6 324.9 333.9 267.5 151.6 904.8 12 1188.79
331.74 426.88 333.01 217.84 1510.1 15 2040.38 443.36 450.76 450.76
391.22 2223.65 19 2851.36 773.88 989.52 737.22 287.96 4587.2 21
3876.85 1022.48 1220.44 976.75 490.76 3279.72
[0247] In this Example, MB49 cells, which exhibit very high cell
growth rates, were intradermally administered, with a focus on
whether or not eradication occurs in tumors of considerable size
having a tumor diameter of 7-8 mm.
[0248] As a result, a tumor suppressing effect was observed with
ADM alone, but eradication was not observed.
[0249] On the other hand, tumor eradication was observed in one of
the three animals in the ADM 100 .mu.g+HVJ-E 5000 HAU
administration group.
[0250] Hence, the results demonstrated an antitumor effect of HVJ-E
and ADM on MB49 cells.
Example 3
Adriamycin (ADM) Sensitivity of MB49 (Transitional Epithelial
Cancer) Cells
(Method)
[0251] The survival rate of MB49 cells, which depends on adriamycin
(ADM) concentration, was examined by the method described below
using WST-8 assay (cell counting kit-8; Dojindo Laboratories).
[0252] Adriamycin (trade name; Adriacin Injection, Kyowa Hakko
Kogyo Co., Ltd.) was added to 10,000 MB49 cells, and the cells were
prepared as 200 .mu.l of sample solution and sown to a 96-well
plate.
[0253] After 48 hours of cultivation, the supernatant was removed
by WST-8 assay, 100 .mu.l of a cell counting kit solution,
previously diluted 10 fold with RPMI medium, was added, and the
plate was allowed to stand at 37.degree. C. for 1.5 hours.
Absorbance at 450 nm was determined using a microplate reader.
Assuming the absorbance of a sample of MB49 cells only to be 100%
survival rate, the survival rates at various concentrations were
calculated. Concentrations that produced survival rates of 50% and
90% were calculated and designated as LD.sub.50 and LD.sub.90.
(Results)
TABLE-US-00007 [0254] ADM (.mu.g/ml) 0.002 0.004 0.008 0.016 0.032
0.063 survival 68.9 60.1 54 48 47.4 34.9 rate (%) ADM (.mu.g/ml)
0.125 0.25 0.5 1 2 survival 27.6 22.6 17.2 11.7 6.7 rate (%)
[0255] The LD.sub.50 concentration of ADM for MB49 cells was
determined to be 0.008 .mu.g/ml and the LD.sub.90 concentration was
determined to be 1 .mu.g/ml (see FIG. 12).
[0256] From this result, it was shown that in the concurrent
administration of MB49 cells, HVJ-E, and ADM into the bladder, the
ADM sensitivity of MB49 cells could be reflected by an in vitro
system.
Example 4
[0257] Antitumor effect of concurrent administration of MB49 cells,
HVJ-E, and ADM into the bladder
(Method)
[0258] Three 6-week-old female C57BL/6Cr Slc mice per group were
anesthetized by administering a mixture of 90 .mu.l of ketamine (50
mg) and 10 .mu.l of xylazine (2% solution) to the left femoral
muscle, a 24G catheter was inserted into the bladder, 100 .mu.l of
each of the following sample solutions was administered using a
1-ml syringe, and the catheter was indwelled for 1 hour.
[0259] The groups established are as follows: [0260] 1: MB49+ADM
(0.008 .mu.g/ml) [0261] 2: MB49+ADM (0.008 .mu.g/ml)+HVJ-E (5000
HAU) [0262] 3: MB49+ADM (1 .mu.g/ml) [0263] 4: MB49+ADM (1
.mu.g/ml)+HVJ-E (5000 HAU)
[0264] The number of MB49 cells administered was 5.times.10.sup.6
cells. After adjustment to a final volume of 100 .mu.l, each
solution was administered into the bladder. Two weeks later, the
bladder was extirpated, and sections were prepared and stained with
HE.
(Results)
[0265] Tumor infiltration was evaluated from images of HE staining.
In Group 1 and Group 2, all animals showed evidence of infiltration
of MB49 cells (see FIGS. 13 and 15). In FIG. 13, the densely purple
(black) portion at the center indicates cancer cells. Even in Group
3, in which the ADM concentration was raised and a dose equivalent
to the LD.sub.90 was administered, infiltration of MB49 cells could
not be prevented in 80% of the animals. However, in the group
receiving the same regimen as Group 4, but supplemented with 5000
HAU HVJ-E, there were no animals having infiltration of MB49 cells
(0/5, infiltration prevention rate 100%) (see FIGS. 14 and 15).
[0266] Hence, it was shown that infiltration of MB49 cells in the
bladder could not be prevented with ADM alone but could be
prevented using ADM and HVJ-E in combination.
[0267] In particular, in the group receiving HVJ-E in combination
with ADM (1 .mu.g/ml), the MB49 take rate in the bladder (cancer
incidence rate) was remarkably suppressed compared with the group
supplemented with ADM alone.
Example 5
Antitumor Effect of Cisplatin (CDDP) and HVJ-E in Combination
(Method)
[0268] CT-26 mouse colon cancer cells were intradermally inoculated
into the backs of BALB/c mice at 5.times.10.sup.6 cells/head. Five
days after the inoculation, treatment with HVJ-E and CDDP was
started in the following animal groups. For each animal group,
tumor volume was calculated as major diameter.times.minor
diameter.times.minor diameter/2.
Animal group: [0269] #1: Physiological saline group [0270] #2:
HVJ-E alone [0271] #3: CDDP 1 mg/kg [0272] #4: CDDP 3 mg/kg [0273]
#5: CDDP 1 mg/kg+HVJ-E (5,000 HAU) [0274] #6: CDDP 3 mg/kg+HVJ-E
(5,000 HAU) [0275] (n=5 or 6 for each group)
(Results)
[0276] The mean values of tumor volume for individual groups tested
using egg- and cell-derived HVJ-E are shown in FIG. 16
(egg-derived) and FIG. 17 (cell-derived). Regarding tumor volume,
no major difference was observed among the three groups:
physiological saline group, HVJ-E-alone administration group, and
CDDP 1 mg/kg administration group [FIG. 16 (egg-derived)]. In the
cell-derived HVJ-E administration group, the regression effect was
greater in the order of the physiological saline group, HVJ-E-alone
administration group, and CDDP 1 mg/kg administration group.
[0277] The tumor regression effect to note in this study was
observed in 33-40% of the animals in the CDDP 1 mg/kg+HVJ-E 5,000
HAU administration group and the CDDP 3 mg/kg+HVJ-E 5,000 HAU
administration group (in both egg- and cell-derived HVJ-E). Hence,
it was confirmed that an antitumor effect was obtained only when an
anticancer drug was simply mixed with HVJ-E without being
encapsulated therein.
Example 5
Responses of HVJ-E and HVJ to Mouse Dendritic Cells (DC)
(Method)
[0278] Recovery of Dendritic Cells (DC) from Mouse
[0279] The femurs and tibias of both lower limbs of a mouse were
collected. Muscles and fats associated with the bones were removed
to the maximum possible extent. Both ends of each bone were cut
off. Serum-free medium (RPMI1640, containing antibiotics) was
injected from the opening using a syringe with a 27G needle to
purge out the bone marrow by pressure. A sample solution containing
the myelocytes recovered by the treatment was passed through a cell
strainer having a pore diameter of 40 .mu.m (BD Falcon Company),
and the debris was removed. Centrifugation was performed at 1,500
rpm for 5 minutes, and the cells were recovered. The cells were
suspended in DC medium [PRMI1640, 10% FCS (MBL, EQITECH Company),
10 ng/500 ml GMCSF (R&D System Company), 2 .mu.l/500 ml
2-mercaptoethanol] (hereinafter DC medium). All the cells were
counted, DC medium was added to obtain a cell density of
1.times.10.sup.6 cells/ml, and the cells were sown to a 24-well
plate at 1 ml per well. Two and four days later, the supernatant
was exchanged with a fresh supply. The floating cells were
recovered 6 days later.
Co-Cultivation of DC and HVJ or HVJ-E
[0280] The amount of cytokines induced by co-cultivation of DC and
the live virus HVJ or inactivated HVJ-E was measured. HVJ or HVJ-E
was added to the culture broth so that the m.o.i. relative to the
DC (1.times.10.sup.6 cells/ml) would be 20. Effects on the presence
or absence of N-tosyl-L-phenylalanine chloromethylketone (TPCK, 15
.mu.M, Wako Pure Chemical Industries), which is an inhibitor of
NF-KB, was also evaluated. Furthermore, the effect of the
Escherichia coli cell wall (lipopolusaccaride, LPS, 10 .mu.M),
which is a typical Th1 immunity induing substance, was also
evaluated. Cultivation was performed for 2 days, and the
supernatant was recovered. The concentrations of the cytokines
IL-12, IL-6, IL-5 and IL-4 in the solution were determined using an
ELISA Development kit from R&D system Company.
[0281] Furthermore, DC maturation was also evaluated.
(Results)
[0282] The results are shown in FIG. 18. Comparing HVJ and HVJ-E,
the amount of IL-12 induced decreased considerably, from 4928 pg/ml
with HVJ stimulation to 748 pg/ml with HVJ-E stimulation. On the
other hand, the amount of IL-6 induced was 1066 pg/ml with HVJ
stimulation and 496 pg/ml with HVJ-E stimulation. For IL-4 and -5,
almost no induction was observed. (FIG. 18)
[0283] Stimulation with HVJ-E and HVJ induced DC maturation to an
extent comparable to that obtained by stimulation with LPS.
Example 6
Effects of HVE-J on Regulatory T Cells (Reg T)
(Method)
[0284] CT-26 (1.times.10.sup.5 cells) was sown to a 96-well plate
in a volume of 200 .mu.l (DMEM, 10% FCS contained). On the
following day, dendritic cells (1.times.10.sup.5 cells) were added.
The medium was exchanged with 200 .mu.l of DC medium. At that time,
HVJ-E was added or not (10.sup.4-10.sup.9/well), and CDDP (1
.mu.g/ml or 3 .mu.g/ml) was also added. On the following day,
5.times.10.sup.5 splenocytes recovered from BALB/c mouse were
added. Cultivation was performed for 3 days. The cells were
recovered, and T cells and regulatory T cells (reg T) were analyzed
for BrdU uptake ratio by FACS.
(Results)
[0285] When HVJ-E, HVJ-E+CDDP (1 .mu.g/ml), and HVJ-E+CDDP (3
.mu.g/ml) were allowed to act, the cell growth of T cells as a
whole was not influenced but the cell growth of regulatory T cells
(reg T) was suppressed HVJ-E-concentration-dependently. (FIGS. 19
and 20)
INDUSTRIAL APPLICABILITY
[0286] Embodying the present invention enables a new chemotherapy
using an HVJ-E vector for all solid cancers rapidly increasing in
Japan, such as lung cancer, breast cancer, digestive cancers such
as stomach cancer, colon cancer, and esophagus cancer, head and
neck cancers (maxillary cancer, tongue cancer, lip cancer, pharynx
cancer, larynx cancer, mouth cancer and the like), gynecological
cancers (uterine cancer, ovarian cancer, uterine cervix cancer and
the like), urological cancers (prostate cancer, bladder cancer,
kidney cancer, renal pelvic and ureteral cancer, testicular tumor,
adrenal tumor, penis cancer and the like), osteochondrosarcoma,
malignant lymphoma, cancer unknown primary, and the like.
Accordingly, the present invention provides a pharmaceutical
composition comprising a chemotherapeutic encapsulated in a viral
vector that induces antitumor immunity, whereby various cancers can
be treated, while increasing antitumor immunity in a living
organism, and suppressing side-effects to normal cells.
[0287] Also, a pharmaceutical composition comprising HVE-J and an
anticancer drug, which is another embodiment of the present
invention, particularly enables efficient treatment for urological
cancer.
[0288] This application is based on patent application Nos.
2004-108599, 2004-136756 and 2005-044639 filed in Japan, the
contents of which are incorporated in full herein by this
reference.
* * * * *
References